E3 ubiquitin ligases are key enzymes within the ubiquitin proteasome system which catalyze the ubiquitination of proteins, targeting them for proteasomal degradation. E3 ligases are gaining importance as targets to small molecules, both for direct inhibition and to be hijacked to induce the degradation of non-native neo-substrates using bivalent compounds known as PROTACs (for ‘proteolysis-targeting chimeras’). We describe Homo-PROTACs as an approach to dimerize an E3 ligase to trigger its suicide-type chemical knockdown inside cells. We provide proof-of-concept of Homo-PROTACs using diverse molecules composed of two instances of a ligand for the von Hippel-Lindau (VHL) E3 ligase. The most active compound, CM11, dimerizes VHL with high avidity in vitro and induces potent, rapid and proteasome-dependent self-degradation of VHL in different cell lines, in a highly isoform-selective fashion and without triggering a hypoxic response. This approach offers a novel chemical probe for selective VHL knockdown, and demonstrates the potential for a new modality of chemical intervention on E3 ligases.
Developing
PROTACs to redirect the ubiquitination activity of E3
ligases and potently degrade a target protein within cells can be
a lengthy and unpredictable process, and it remains unclear whether
any combination of E3 and target might be productive for degradation.
We describe a probe-quality degrader for a ligase–target pair
deemed unsuitable: the von Hippel–Lindau (VHL) and BRD9, a
bromodomain-containing subunit of the SWI/SNF chromatin remodeling
complex BAF. VHL-based degraders could be optimized from suboptimal
compounds in two rounds by systematically varying conjugation patterns
and linkers and monitoring cellular degradation activities, kinetic
profiles, and ubiquitination, as well as ternary complex formation
thermodynamics. The emerged structure–activity relationships
guided the discovery of VZ185, a potent, fast, and selective degrader
of BRD9 and of its close homolog BRD7. Our findings qualify a new
chemical tool for BRD7/9 knockdown and provide a roadmap for PROTAC
development against seemingly incompatible target–ligase combinations.
Proteolysis targeting
chimeras (PROTACs) are catalytic heterobifunctional
molecules that can selectively degrade a protein of interest by recruiting
a ubiquitin E3 ligase to the target, leading to its ubiquitylation
and degradation by the proteasome. Most degraders lie outside the
chemical space associated with most membrane-permeable drugs. Although
many PROTACs have been described with potent activity in cells, our
understanding of the relationship between structure and permeability
in these compounds remains limited. Here, we describe a label-free
method for assessing the permeability of several VH032-based PROTACs
and their components by combining a parallel artificial membrane permeability
assay (PAMPA) and a lipophilic permeability efficiency (LPE) metric.
Our results show that the combination of these two cell-free membrane
permeability assays provides new insight into PROTAC structure–permeability
relationships and offers a conceptual framework for predicting the
physicochemical properties of PROTACs in order to better inform the
design of more permeable and more effective degraders.
Inducing
post-translational protein knockdown is an important approach
to probe biology and validate drug targets. An efficient strategy
to achieve this involves expression of a protein of interest fused
to an exogenous tag, allowing tag-directed chemical degraders to mediate
protein ubiquitylation and proteasomal degradation. Here, we combine
improved HaloPROTAC degrader probes with CRISPR/Cas9 genome editing
technology to trigger rapid degradation of endogenous target proteins.
Our optimized probe, HaloPROTAC-E, a chloroalkane conjugate of high-affinity
VHL binder VH298, induced reversible degradation of two endosomally
localized proteins, SGK3 and VPS34, with a DC50 of 3–10
nM. HaloPROTAC-E induced rapid (∼50% degradation after 30 min)
and complete (Dmax of ∼95% at 48
h) depletion of Halo-tagged SGK3, blocking downstream phosphorylation
of the SGK3 substrate NDRG1. HaloPROTAC-E more potently induced greater
steady state degradation of Halo tagged endogenous VPS34 than the
previously reported HaloPROTAC3 compound. Quantitative global proteomics
revealed that HaloPROTAC-E is remarkably selective inducing only degradation
of the Halo tagged endogenous VPS34 complex (VPS34, VPS15, Beclin1,
and ATG14) and no other proteins were significantly degraded. This
study exemplifies the combination of HaloPROTACs with CRISPR/Cas9
endogenous protein tagging as a useful method to induce rapid and
reversible degradation of endogenous proteins to interrogate their
function.
Bivalent PROTACs work drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhanced degradation. Here, we designed trivalent PROTACs consisting of a bivalent BET inhibitor and an E3 ligand, tethered via a branched linker. We identified VHL-based SIM1 as a low picomolar BET degrader, with preference for BRD2. Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anti-cancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable
in vivo
pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.
Stabilizing biomolecular interactions with synthetic molecules to impact biological function is a concept of enormous appeal. Although much effort has been devoted to disrupting proteinprotein interactions (PPIs) using inhibitors, less attention has been directed toward the reverse strategy, that of inducing new PPIs. However recent years have seen a resurgence of interest in designing molecules that bring proteins together. This approach promises to significantly expand the range of tractable targets for chemical biology and therapeutic intervention. Pioneering structural and biophysical investigation of ternary complexes formed by mono-and bifunctional ligands highlight that proximity-induced stabilization or de novo formation of PPIs are a common feature of their molecular recognition. In this review, we illustrate these concepts and advances with representative case studies, and highlight progress over the past three years, with particular focus on recruitment to E3 ubiquitin ligases by "molecular glues" and chimeric dimerizers (PROTACs) for targeted protein degradation.
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